Kir2.1 channel regulates macrophage polarization via the Ca²⁺/CaMK II/ERK/NF-κB signaling pathway

Macrophages are the major component of the innate immune system and play an important role in preventing microbial invasion, inflammatory response, tissue remodeling and repair (Biswas and Mantovani, 2010; Gordon and Martinez, 2010; Murray et al., 2014). Macrophages can recognize danger signals through receptors and initiate specialized activation programs. There are two ways of macrophage activation: classic activation and alternative activation. The classic activation can be induced by lipopolysaccharides (LPS), also called M1 activation, and is associated with the proinflammatory response required to recruit more immune cells and kill invading pathogens. The alternative activation can be induced by interleukin-4 (IL-4) in vitro, called M2 activation, in which M2 macrophages are involved in tissue repair and the resolution of inflammation (Mantovani et al., 2005; Martinez et al., 2008). Many studies have shown that M1 activation is the predominant phenotype in the early stage of inflammation, whereas after the acute inflammation phase, M2 macrophages increase gradually and play a more important role in the process. The prognosis of inflammation is largely determined by the balance of M1 and M2 macrophages; for example, the cardiac remodeling after myocardial infarction is closely associated with macrophage polarization (Li et al., 2021; Odegaard et al., 2007). Thus, emphasis on macrophage polarization research might lead to a new potential therapeutic target. Until now, many transcription factors have been found to participate in the process of macrophage polarization, such as the signal transducer and activator of transcription (STAT) protein family, peroxisome proliferator-activated receptor-γ (PPARγ, encoded by PPARG), cAMP response element binding protein (CREB)-CCAAT/enhancer binding protein (C/EBP), hypoxia-inducible factors (HIFs) and NF-κB (Li et al., 2021; Luan et al., 2015; Odegaard et al., 2007; Xue et al., 2014). However, the underlying mechanisms of macrophage polarization remain unclear.

In recent years, many functional ion channels have been discovered in macrophages, including transient receptor potential (TRP) channels and K⁺ channels. These channels play a role in phagocytosis (Link et al., 2010), cell survival (Serafini et al., 2012), proliferation and activation (Schilling et al., 2014; Vicente et al., 2003), and cytokine production (Yamamoto et al., 2008). Several publications reported the roles of the TRPM7 channel in LPS-induced macrophage activation, macrophage polarization, and macrophage-mediated immunomodulation (Li et al., 2020; Qiao et al., 2021; Schappe et al., 2018). Kv1.3 (or KCNA3) and TWIK2 (or KCNK6) channels were found to fulfill an important role in NLRP3 inflammasome-induced inflammation (Di et al., 2018; Mei et al., 2019). Thus, ion channels can be regarded as potential therapeutic targets in many immune diseases (Eder, 2010). The cell resting membrane potential is a result of a balance between inward and outward ionic currents, and the membrane potential has been reported to be involved in multiple normal physiological processes, such as cell survival, proliferation and differentiation, wound healing and tissue regeneration. Further studies revealed the possible signaling pathways that respond to membrane potential; the K-RAS/MAPK, Ca²⁺/calcineurin/MEF2 and PI3Kγ/PTEN signaling pathways could be activated or inhibited upon changes in membrane potential (depolarization or hyperpolarization) (Konig et al., 2006, 2004; Zhao et al., 2006; Zhou et al., 2015). In 2016, the role of membrane potential in the regulation of macrophage polarization was documented, but the underlying mechanism was unclear (Li et al., 2016). Kir2.1 (or KCNJ2) belongs to the inwardly rectifying K⁺ (K_ir) channel family, which allows more K⁺ ions to flow into the cell rather than out of the cell. Each subunit of the Kir2.1 channel protein only has two transmembrane segments. Kir2.1 is the most important channel that generates and maintains polarized membrane potential, which is the basis for excitability generation of excitable cells (Hibino et al., 2010; Lopatin and Nichols, 2001). However, interestingly, the Kir2.1 channel is also expressed in macrophages, such as peritoneal macrophages (Gallin and Livengood, 1980) and microglia, which have a hyperpolarized membrane potential. There is also evidence that the Kir2.1 channel is necessary for microglial migration (Lam and Schlichter, 2015). Another study reported the expression of the Kir2.1 channel was changed in macrophages treated by LPS or TNFα (encoded by TNF), indicating the involvement of Kir2.1 in macrophage activation (Vicente et al., 2003). However, how the Kir2.1 channel takes part in macrophage activation and/or polarization and whether the roles of the Kir2.1 channel in macrophages are related to membrane potential are not clear. We designed experiments to explore the role of the Kir2.1 channel in regulating macrophage polarization and its underlying mechanisms.

In this study, we found that inhibition of the Kir2.1 channel prevented M1 polarization but promoted M2 polarization of RAW264.7 macrophages. LPS-induced M1 polarization was also suppressed by a depolarized membrane potential induced with 70 mM K⁺ solution. The decrease of Ca²⁺ influx caused by depolarized membrane potential (blockade of Kir2.1 channel or 70 mM K⁺) inhibited the CaMK II/ERK/NF-κB signaling pathway, leading to suppression of macrophage M1 polarization. In vivo studies showed that LPS-induced peritonitis could be improved by treatment with the Kir2.1 channel blocker. Our novel findings demonstrate the important role of the Kir2.1 channel in macrophage polarization, and suggest that the Kir2.1 channel could be a potential target for modulating inflammation.

First, we recorded the inwardly rectifying K⁺ current (I_K1) in RAW264.7 cells using the classic step stimulation protocol and specific blocker (Ba²⁺). Ba²⁺ is the most classic and commonly used blocker of Kir2.1 current (Li et al., 2009). Fig. 1A shows a representative inwardly rectifying current recorded with 200-ms voltage steps to potentials between −140 and +50 mV from a holding potential of −40 mV in the absence and presence of 0.5 mM Ba²⁺. The current was significantly suppressed by Ba²⁺. Fig. 1B displays the current–voltage (I–V) relationships of the Ba²⁺-sensitive current in the cells treated with or without LPS (100 ng/ml) or IL-4 (20 ng/ml). There were no significant differences in current densities before and after stimulation with LPS or IL-4 (Fig. 1C, P=0.878 and 0.622, respectively, n=6–8). Fig. 1D shows that the gene expression of Kir2.1 is much higher than that of other K⁺ channels that also contribute to setting up the membrane potential, suggesting that the recorded Ba²⁺-sensitive current in RAW264.7 cells is mainly mediated by the Kir2.1 channel. Quantitative reverse transcription PCR (qRT-PCR) and western blot results also showed no remarkable differences in the gene and protein levels of Kir2.1 in the cells before and after treatment with LPS or IL-4 (Fig. 1E, P=0.532 and 0.669, respectively; Fig. 1F, P=0.735 and 0.283, respectively; n=3 samples in each group).

To explore the role of the Kir2.1 channel in macrophage polarization, we tested the expression of M1- and M2-related polarity markers in RAW264.7 cells treated with LPS or IL-4, and with either 3 μM ML133, a specific Kir2.1 channel blocker (Wang et al., 2011) or 0.5 mM Ba²⁺; or in cells infected with Ad-mKir2.1 shRNA virus treated with LPS or IL-4. Fig. 2A shows the significant decrease of Kir2.1 gene expression in cells infected with the Ad-mKir2.1 shRNA virus (**P=0.0044, 0.0061 and 0.0032 for shRNAs #1, #2 and #3, respectively, versus scrambled; n=3). qRT-PCR results showed that the expression levels of the M1 markers IL-1β (encoded by IL1B), IL-6, iNOS (NOS2), and TNFα were significantly decreased in cells treated with LPS and the Kir2.1 blocker ML133, and in cells treated with LPS and Ad-mKir2.1 shRNA virus, compared to cells treated with only LPS (Fig. 3A; IL-1β: **P<0.0001; IL-6: **P=0.00013 and 0.00054 for ML133 and Ba²⁺, respectively; iNOS: **P<0.0001; TNFα: **P<0.0001; significance was calculated for results from cells treated with LPS + ML133 or LPS + Ba²⁺, versus those treated with LPS alone; n=5 or 7. Fig. 2B, IL-1β: **P=0.0008; IL-6: **P<0.0001; iNOS: **P<0.0001; TNFα: *P=0.042; for Kir2.1 shRNA + LPS versus scrambled shRNA + LPS; n=3). Although the expression of the M2 markers Arg1, CD206 (MRC1), IGF-1 and IL-10 were remarkably increased in cells treated with IL-4 and the Kir2.1 blocker ML133 or with IL-4 and Ad-mKir2.1 shRNA virus compared to cells treated only with IL-4 (Fig. 3D; Arg1: **P=0.0012 and **P<0.0001; CD206: **P=0.00093 and **P<0.0001; IGF-1: **P=0.0014 and 0.0024; IL-10: **P=0.0022 and 0.0013; for IL4 + ML133 or IL4 + Ba²⁺ treatment versus IL-4 treatment alone, n=4. Fig. 2C; Arg1: **P=0.00014; CD206: **P=0.0093; IGF-1: **P=0.0013; IL-10: **P=0.00074; for Kir2.1 shRNA + LPS versus scrambled shRNA + LPS; n=3). Flow cytometry was used to measure the percentage of polarized macrophages; CD86 and CD206 are classic surface markers of M1 and M2 macrophage, respectively. Compared to cells that were treated with LPS alone, the proportion of CD86⁺ cells was much lower for cells treated with LPS+ML133 (Fig. 3B,C; *P=0.022; n=3); Fig. 3E displays representative results of flow cytometry analyses of untreated, IL-4-treated and IL-4+ML133-treated RAW264.7 cells. Compared to IL-4-treated cells, the proportion of CD206⁺ cells was much higher for cells treated with IL-4+ML133 (Fig. 3F; *P=0.031; n=3). These results are consistent with the qRT-PCR results, indicating that blocking Kir2.1 channel inhibits M1 polarization and promotes M2 polarization of RAW264.7 cells.

To explore how the Kir2.1 channel is involved in macrophage polarization, Ca²⁺ imaging was employed to measure the changes in intracellular Ca²⁺. Fig. 4A shows the store-operated Ca²⁺ entry in normal RAW264.7 cells; the intracellular background Ca²⁺ levels were very low when the cells were incubated in a Ca²⁺-free solution, whereas a significant increase of intracellular Ca²⁺ levels could be observed upon the addition of a 2 mM Ca²⁺ solution. Fig. 4B shows that compared to untreated RAW264.7 cells, more Ca²⁺ ions influx in cells treated with LPS. Store-operated Ca²⁺ entry was almost completely suppressed by treatment with the Kir2.1 channel blocker ML133 (3 μM), which was added after or coincident with the addition of 2 mM Ca²⁺ (Fig. 4C,D). Moreover, store-operated Ca²⁺ entry in LPS-treated RAW264.7 cells was also suppressed by ML133 treatment (Fig. S1A). In order to confirm the effects of Kir2.1 on store-operated Ca²⁺ entry, we found that the level of Ca²⁺ entry was affected in RAW264.7 cells transfected with Kir2.1-shRNA (Fig. S1B). Fig. 4E shows that Ca²⁺ entry in cells treated with 5 mM K⁺ + 2 mM Ca²⁺ solution was suppressed significantly when the extracellular solution was changed to a 70 mM K⁺+2 mM Ca²⁺ solution; and, subsequently, the Ca²⁺ influx was partially recovered upon treatment with a 70 mM K⁺+5 mM Ca²⁺ solution. The original representative images at the corresponding time points in these solutions are shown on the right in Fig. 4E. These data suggest that the Kir2.1 channel participates in store-operated Ca²⁺ entry via controlling membrane potential in RAW264.7 cells.

It is well known that Kir2.1 is the major contributor towards the generation of membrane potential. According to the Nernst equation, the membrane potential will depolarize in a solution with high K⁺ concentration (Powell and Brown, 2021). Here, we wanted to verify whether regulation of the Kir2.1 channel in macrophage polarization is associated with the membrane potential. Fig. 5 shows that the expressions of M1-related markers were decreased in a K⁺ concentration-dependent manner. It was interesting to find that these reduced expressions of the M1 markers were partially restored by increasing Ca²⁺ concentration to 5 mM in the 70 mM K⁺ solution (IL-1β: **P<0.0001, ^##P=0.00062; IL-6: **P=0.00051, ^#P=0.033; iNOS: **P=0.0047, ^##P=0.0096; TNFα: **P=0.0012, ^##P=0.0045; n=3. ‘**’ indicates P-values for samples compared to those treated with LPS + 5 mM K⁺ solution, ‘^#’ and ‘^##’ indicate P-values for samples compared to those treated with LPS+2 mM Ca²⁺ + 70 mM K⁺).

Next, we tested the possible signaling pathway by which the Kir2.1 channel regulated macrophage polarization. The results above suggest that the Kir2.1 channel is involved in store-operated Ca²⁺ entry. Increased intracellular Ca²⁺ can activate CaMK II, phosphorylated CaMK II can activate ERK1/ERK2 (encoded by MAPK3 and MAPK1, respectively; collectively referred to as ERK1/2 or ERK), which are upstream signal molecules of NF-κB (Ding et al., 2019). It is well known that NF-κB is a key molecule in the control of macrophage polarization (Wang et al., 2021). Experiments were designed to demonstrate the hypothesis above; Fig. 6A shows a representative experiment. Compared to the negative control, high levels of TLR4, phospho (p)-CaMK II, p-ERK1/2 and p-NF-κB proteins were detected in RAW264.7 cells treated with LPS for 6 h. Except for TLR4, the protein levels of p-CaMK II, p-ERK and p-NF-κB were decreased remarkably when RAW264.7 cells were treated with LPS+ML133 or LPS+70 mM K⁺; this decrease could partially be restored after additional Ca²⁺ was added to the LPS+70 mM K⁺ solution. The relative values of the protein levels of p-CaMK II, p-ERK and p-NF-κB in cells incubated with LPS in 5 mM K⁺, LPS in 5 mM K⁺+ML133, LPS in 70 mM K⁺ and LPS in 70 mM K⁺+5 mM Ca²⁺ solutions are shown (Fig. 6B; *P<0.05 and **P<0.01 for samples versus LPS treatment alone; ^#P<0.05 for samples versus LPS + 70 mM K⁺; n=3). In addition, the protein expression of p-STAT6, a key signaling molecular of M2 polarization, was analyzed. As expected, ML133 or 70 mM K⁺ treatment promoted the expression of p-STAT6 (Fig. S2A). These data indicate that the Kir2.1 channel might regulate macrophage polarization through the Ca²⁺/CaMK II/ERK/NF-κB and p-STAT6 signaling pathways, which appears to be independent of TLR4.

We examined the importance of the Kir2.1 channel in macrophage polarization in vivo by challenging mice with intraperitoneal injections of LPS (0.4 mg/kg) or LPS+ML133 (1 mg/kg) (Fig. 7A). After LPS administration, mice were observed for pathological symptoms by using a composite clinical score (Table S2) (Schappe et al., 2018); the higher the score, the more serious the peritonitis. Scores were recorded in a double-blinded manner. There was a significant decrease in the composite clinical score in mice treated with LPS+ML133 compared to that of mice treated with LPS alone, suggesting that blocking the Kir2.1 channel effectively alleviated peritonitis (Fig. 7B; **P=0.0091 for LPS+ML133 versus LPS, n=5 mice). 4 h after LPS challenge, peritoneal macrophages were collected to perform qRT-PCR analysis. Fig. 7C–F shows that the gene expressions of the M1 markers TNFα, IL-1β, iNOS and IL-6 were increased two- to five-fold after LPS administration, whereas in the mice administrated with LPS+ML133, the expression levels of the these four genes were remarkably decreased (by ∼20% and ∼60%, respectively, Fig. 7C–F, **P<0.001 for LPS+ML133 versus LPS, n=5 mice). Then, flow cytometry was used to examine macrophages from mice treated with LPS or LPS+ML133 for 24 h (Fig. 7G). Peritoneal macrophages were marked with the F4/80 antibody, the percentage of macrophages in each group was about 10%. Subsequently, the CD86 antibody was used to mark M1 macrophages. Compared to LPS-treated mice, the proportion of CD86⁺ peritoneal macrophages was much lower in mice treated with LPS+ML133 (Fig. 7H, **P=0.0011 for LPS+ML133 versus LPS, n=5 mice).

In the present study, we provide solid evidence to understand the pivotal role of the Kir2.1 channel in the regulation of macrophage polarization via the Ca²⁺/CaMK II/ERK/NF-κB signaling pathway, which is summarized in Fig. 8. Our in vivo experiments show that the Kir2.1 channel blocker effectively alleviates LPS-induced peritonitis by inhibiting M1 macrophage polarization.

About 40 years ago, an inward rectification current was recorded in mouse macrophages (Gallin and Livengood, 1980, 1981; Gallin and Sheehy, 1985), which can be inhibited by barium chloride. Since then, Kir2.1 current and protein expression were detected in many kinds of macrophages, including microglial cells (Schappe et al., 2018), bone marrow-derived macrophages (BMDMs) (Vicente et al., 2003) and THP-1 macrophages (Qiao et al., 2021). Further studies suggest that the Kir2.1 channel might participate in macrophage maturation and foam-cell formation (Di et al., 2018), and mediate microglial proliferation and migration (Lam and Schlichter, 2015; Li et al., 2009; Mei et al., 2019). Although the involvement of the Kir2.1 channel in macrophages activation has been reported, very little is known about the role of the Kir2.1 channel in macrophage polarization and the underlying mechanisms. Here, we find that the inhibition of the Kir2.1 channel strongly suppresses M1 polarization and promotes M2 polarization that are induced by LPS and IL-4, respectively. To exclude possible non-specific effects of the inhibitors, shRNA was used to further confirm the effect of the Kir2.1 channel on macrophage polarization, which gave similar results. Interestingly, in our study, there were no significant changes of Kir2.1 channel expression during macrophage polarization. This result is supported by some publications; for example, no significant changes in Kir2.1 current were observed in microglial cells before and after treatment with LPS or IL-4 (Di Lucente et al., 2018; Lam and Schlichter, 2015; Nguyen et al., 2017). However, other studies have shown that the Kir2.1 current is significantly suppressed in BMDMs after treatment with LPS (Moreno et al., 2013), whereas in THP-1 macrophages treated with phorbol 12-myristate 13-acetate (PMA), the Kir2.1 current is remarkably activated (DeCoursey et al., 1996). In addition, the Kir2.1 current is inhibited in the process of macrophage maturation (Powell and Brown, 2021). These inconsistent results indicate that the role of Kir2.1 in macrophages is controversial and might differ between cell types.

Ca²⁺ is a very important second messenger in cells, and Ca²⁺-related signaling pathways participate in multiple cellular physiological processes. In non-excitable cells, many channels or receptors are involved in intracellular Ca²⁺ homeostasis, such as TRP channels, Ca²⁺ release-activated Ca²⁺ channels (STIM, ORAI1), ryanodine receptor, IP3 receptor, Ca²⁺ pump and Na⁺-Ca²⁺ exchanger, which make up a complex network to regulate the intracellular Ca²⁺ concentration. In macrophages, studies show that intracellular Ca²⁺ regulates the production of inflammatory factors and phagocytosis (Watanabe et al., 1996). The involvement of Ca²⁺ in LPS-induced macrophage activation has been well documented. A recent study reported that Ca²⁺ influx mediated by TRPM7 is required for LPS-induced macrophage activation, and the translocation of NF-κB and IRF3 is also dependent on TRPM7-mediated Ca²⁺ influx (Schappe et al., 2018). TRPV4-mediated Ca²⁺ influx contributes to LPS-induced macrophage activation via the calcineurin/NFATc3 pathway in lung injury and lung inflammation (Li et al., 2019). Studies from microglia BV2 cells showed that increased intracellular Ca²⁺ could cause the phosphorylation of CaMKIIα, ERK1/2 and NF-κB (p65) (Lu et al., 2017). The contribution of intracellular Ca²⁺ to M1 macrophage polarization has been reported (Di Lucente et al., 2018), but the underlying mechanism is not clear. In our study, we also observed the increase of Ca²⁺ influx in macrophages treated with LPS, and the increased intracellular Ca²⁺ could activate the CaMKII/ERK/NF-κB signaling pathway, which might determine macrophage polarization.

As mentioned above, there are several pathways for Ca²⁺ influx; however, Ca²⁺ influx is not always possible because Ca²⁺ channels are almost closed in their resting states. A hyperpolarized membrane potential generated by Kir2.1 is regarded as a strong driving force for Ca²⁺ influx. Kir2.1 was found to be the predominant K⁺ channel in RAW264.7 cells in our study. Several studies demonstrated the contribution of the Kir2.1 channel to Ca²⁺ influx. Qi et al. (2015) reported that the upregulation of the Kir2.1 channel hyperpolarized the membrane potential, increasing intracellular Ca²⁺ via store-operated Ca²⁺ entry, thereby promoting fibroblast proliferation in dog hearts with atrial fibrillation. Another study shows that Kir2.1-controlled Ca²⁺ entry triggered human myoblast differentiation via the activation of the calcineurin pathways (Konig et al., 2006). Interestingly, the activation state of microglia was also determined by Kir2.1 channel-mediated Ca²⁺ influx via the Ca²⁺ release-activated Ca²⁺ (CRAC) channel (Lam and Schlichter, 2015). Hyperpolarization also can increase cytoplasmic Ca²⁺ in arteriolar endothelial cells (Dora and Garland, 2013). Thus, we focused on store-operated Ca²⁺ entry in macrophages. Blocking the Kir2.1 channel with the inhibitor ML133 significantly suppressed Ca²⁺ entry. High K⁺ solutions were employed to confirm the contribution of Kir2.1 to Ca²⁺ influx by establishing a hyperpolarized membrane potential. Meanwhile, Ca²⁺ entry could partially be restored by increasing Ca²⁺ concentration in a solution with high K⁺ concentration. Although Kir2.1 could not be activated in RAW264.7 cells in our study, the membrane potential set up by Kir2.1 is required for supporting Ca²⁺ entry.

In summary, we demonstrated the important role of the Kir2.1 channel in macrophage polarization, and that the Kir2.1 channel blocker effectively alleviated LPS-induced peritonitis by inhibiting M1 macrophage polarization in vivo. The present results indicate that the Kir2.1 channel in macrophages might be a potential therapeutic target for inflammatory diseases.

RAW264.7 cells were purchased from Procell Life Science & Technology with short tandem repeat (STR) authentication (Wuhan, China), and cultured at 37°C and 5% CO₂ with high glucose Dulbecco's Modified Eagle Medium (DMEM, 10-013-CVR, Corning) containing 10% fetal bovine serum (FBS; 16000-044, Gibco), 100 μg/ml penicillin and 100 μg/ml streptomycin (Invitrogen, China). The culture medium was changed every 2 days per standard cell culture protocols. 100 ng/ml LPS (Escherichia coli O111:B4, L2630, Sigma-Aldrich) or 20 ng/ml IL-4 (SRP3093, Sigma-Aldrich) were used to stimulate RAW264.7 cells for 6 h or 24 h, respectively, based on our pre-experiments and published studies (Schappe et al., 2018).

RAW264.7 cells were cultured in dishes (430166, Corning) used for Kir2.1 current recording. The cells were placed into a cell chamber mounted on the stage of an inverted microscope (Olympus, IX70, Japan), and allowed to settle to the bottom of the cell chamber before being superfused with bath solution (2 ml/min). Whole-cell current was recorded with an Axopatch 200B amplifier and Clampex 10 software (Axon Instruments, USA). Glass electrodes were pulled with a Brown-Flaming puller (Model P-97, Sutter Instrument, CA), and the resistance of the electrodes was 2–3 MΩ when filled with pipette solution. Pipette potentials were zeroed before the pipette contacted the cells. After a gigaohm seal was obtained by negative suction, the cell membrane was ruptured by gentle suction to establish whole-cell configuration. Electrical signal was low-pass filtered at 5 kHz and stored on the hard disk of a computer. All experiments were conducted at room temperature.

The bath solution contained NaCl 140 mM, KCl 5.4 mM, MgCl₂ 1 mM, CaCl₂ 1.8 mM, Glucose 10 mM and HEPES 10 mM; pH adjusted to 7.3 with NaOH. The pipette solution contained K-aspartate 110 mM, KCl 20 mM, Mg-ATP 5 mM, GTP 0.1 mM, HEPES 10 mM, EGTA 5 mM; pH adjusted to 7.2 with KOH. All chemicals were purchased from Sigma-Aldrich. ML133 (1222781-70-5) was dissolved in DMSO and prepared as a 3 or 10 mM stock solution stored at −20°C.

Total RNA was isolated using the TRIzol method (Invitrogen, China). RNA concentration was measured using a NanoDrop 2000 (Thermo Fisher Scientific) and normalized among the experimental sample set. cDNA was generated using the PrimeScript Reverse Transcription (RT) Reagent Kit (Takara, Japan) and used for qRT-PCR. qRT-PCR reactions were prepared with SYBR Premix Ex Taq II (Takara, Japan) using primers specific for Kir2.1, IL-1β, IL-6, iNOS, TNFα, Arg1, CD206, IGF-1 and IL-10 (Table S1). The reactions were run in the LightCycler480 real-time PCR detection system (Roche, USA) according to the manufacturer's instructions. Relative mRNA levels were determined using the ΔΔCt method and normalized to GAPDH levels.

Trypsinized RAW264.7 cells or isolated peritoneal macrophages were washed with PBS, and incubated with the fluorescently conjugated antibodies F4/80-APC (1:10, 17-4801-82), CD86-PE (1:100, 12-0862-82) and CD206-PE (1:100, 12-2061-82) or their respective isotype controls at 4°C for 30 min. Then cells were washed three times with PBS and analyzed using CytoFLEX S flow cytometer (Beckman Coulter). For each sample analyzed, a minimum of 10,000 events were acquired. All antibodies were purchased from eBioscience (CA, USA).

Western blotting was performed using standard protocols Briefly, RAW264.7 cells were lysed with modified RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 2 mM sodium pyrophosphate, 25 mM β-glycerophosphate, 1 mM EDTA, 1 mM Na₃VO₄, 0.5 μg/ml leupeptin) for 30 min on ice, cell lysates were then centrifuged at 12,000 g for 20 min at 4°C. After transferring the supernatant to a new ice-cold tube, protein concentration was quantified using a bicinchoninic acid assay kit. Samples were then mixed with SDS sample buffer, denatured at 95°C for 5 min and resolved with 10% SDS-page gels. Gels were transferred to polyvinylidene difluoride membrane papers, the membranes were blocked with 5% non-fat milk in TBS containing 0.1% Triton X-100 (TTBS) for 1 h. Then membranes were incubated with primary antibodies overnight at 4°C. Anti-CaMK II (1:1000, sc-13141), anti-p-CaMK II (1:800, sc-32289), anti-NF-κB (P65) (1:1000, sc-8008), anti-p-NF-κB (p-P65) (1:500, sc-166748) antibodies were purchased from SantaCruz Biotechnology (CA, USA). Anti-TLR4 (1:400, 66350-1-Ig), anti-ERK (1:500, 16443-1-AP), anti-p-ERK (1:500, 28733-1-AP), anti-Kir2.1 (1:500, 19965-1-AP), anti-β-actin (1:2000, 66009-1-lg), anti-GAPDH (1:2000, 60004-1-Ig) antibodies were purchased from ProteinTech (Wuhan, China). After washing with TTBS, the membranes were incubated with HRP-conjugated secondary antibodies (SA00001-1, SA00001-2, ProteinTech, China) for 1 h. The membranes were washed again with TTBS and then the blots were analyzed using an enhanced chemiluminescence detection system.

The Ca²⁺ imaging was performed using a previously described procedure (Hu et al., 2009). RAW264.7 cells were cultured for 2 h at 37°C with 5 μM Fluo-4 AM (F14201, Invitrogen), 0.02% pluronic acid (9003-11-6, Sigma-Aldrich) and 2.5 mM probenecid (57-66-9, Sigma-Aldrich) in FBS-free DMEM, and then incubated in Ca²⁺-free Tyrode solution for 30 min. The Tyrode solution contains NaCl 140 mM, KCl 5 mM, MgCl₂ 1 mM, CaCl₂ 1.8 mM, HEPES 10 mM, glucose 10 mM, pH was adjusted to 7.3 with NaOH. Fluo-4 AM was excited by a 488 nm argon-ion laser and emission was detected at 530 nm. Cytosolic Ca²⁺ activity was monitored every 10 s using confocal microscopy (Olympus FV300, Tokyo, Japan) at room temperature. To measure store-operated Ca²⁺ entry, cells were first exposed to a Ca²⁺-free solution, the extracellular Ca²⁺ concentration was then increased to 2 mM to measure Ca²⁺ entry via store-operated channels.

For Kir2.1 knockdown, three mouse Kir2.1 shRNA (Kir2.1-shRNA #1, Kir2.1-shRNA #2, Kir2.1-shRNA #3) sequences were synthesized and cloned into the vector pAd-GFP-U6-shRNA (GenePharma, Shanghai, China). The sequences of Kir2.1 shRNAs are as follows: #1, 5′-GGAGCCGCTTTGTGAAGAAAG-3′; #2, 5′-GCCCAATTGCTGTCTTCATGG-3′; and #3, 5′-GGAGTTCGTATCTGGCCAATG-3′. Adenovirus construction, purification and titration were performed by Shanghai GenePharma. Viral particles of Ad-GFP-mKir2.1 shRNA and Ad-GFP control shRNA were obtained at concentrations in the order of 10⁹ PFU/ml. Cells at 60–80% confluence were transduced with adenovirus particles of Ad-GFP-mKir2.1 shRNA or Ad-GFP control shRNA at concentrations of 2×10⁷ PFU/ml.

Six- to 8 week-old male C57BL/6 mice were divided into three groups (control, LPS, LPS+ML133) and were injected intraperitoneally with PBS, 0.4 mg/kg LPS or 0.4 mg/kg LPS+1 mg/kg ML133. Mice were observed for up to 24 h, and peritoneal macrophages were then isolated by injection of 5 ml DMEM containing FBS into the peritoneal cavity. The peritoneal lavage fluid was collected and cells were centrifuged at 4°C for 5 min at 500 g. The cells were collected to perform qRT-PCR, or the pellet was re-suspended in ACK lysis buffer (Beyotime Biotechnology, Wuhan, China) for 5 min to lyse red blood cells and subjected to staining for flow cytometry. All experimental procedures of the study were approved by the Animal Ethics Committee of Ningbo University (Approved NO: 2019-48).

Whole-cell currents were analyzed using Clampfit 10.1 software (Axon Instruments, USA). Western blot bands were measured using Image J. Fluo-4 signals were analyzed using FlowView FV300 (Olympus, Tokyo, Japan). All graphical data are presented as mean±s.d. A two-tailed unpaired Student's t-test was used as appropriate to evaluate the differences between two group means, and one-way analysis of variance was used for multiple groups. Values of P<0.05 were considered to indicate statistical significance.

The peer review history is available online at https://journals.biologists.com/jcs/article-lookup/doi/10.1242/jcs.259544.